The herpesvirus maturational protease is essential for the production of infectious virus and is synthesized as a precursor that has multiple functions during capsid assembly. Since its discovery 12 years ago, it has received considerable attention as a possible target for antivirals, and in the past 7 years a wealth of information about this interesting, albeit challenging, enzyme has accumulated. The 28-kDa enzyme is derived from a 74-kDa precursor by autoproteolytic cleavage, first at the (M)aturational site near its carboxyl end, and then at the (R)elease site. R-site cleavage frees assemblin from the approximately 44-kDa carboxyl portion of the precursor. The proteolytic domain, called assemblin in cytomegaolvirus, has been cloned, purified, and studied in vitro. The enzyme is activated by dimerization but the dimer pair has two separate active sites, moreover, it differs remarkably from other serine proteases by its new fold (7-stranded ( barrel) and catalytic triad (Ser-His-His). No comparable information is available about the precursor. Our objective is to learn more about this viral enzyme through biochemical and genetic studies, with particular attention to its precursor. The specific aims are intended to help accomplish that objective by (i) applying mutant viruses to study the mechanism of this protease during virus infection, (ii) developing a protease mutant whose activity can be switched on in cells and in vitro by chemical rescue, (iii) investigating the ability of two catalytic-site mutants to form a complementation complex with selective specificity for the maturational-cleavage site, (iv) taking advantage of recent findings to investigate the structure of the precursor and compare its enzymatic properties with those of assemblin, and (v) investigating requirements and consequences of dimerization by both forms of the enzyme. Results of this work are anticipated to provide useful new information about this novel member of the serine proteinase family, and contribute to development of inhibitors that will block its function. It is also likely that useful new information will be generated in the areas of viral proteinase mechanisms and herpesvirus replication.